Comparison of diagnostic techniques to detect the clam pathogen Perkinsus olseni

Abstract

Perkinsosis in clams in Galicia (NW Spain) is caused by the protozoan parasite Perkinsus olseni Lester & Davis, 1981. We used 5 clam species of commercial interest cultured in Galicia (Ruditapes decussatus, R. philippinarum, Venerupis pullastra, V. rhomboides, and Donax trunculus) to compare various P. olseni diagnostic techniques. Results of a nested PCR assay for the diagnosis of P. olseni were compared to those obtained using 2 classical methods of diagnosis proposed by the World Organisation for Animal Health (OIE), viz. histology and incubation in Ray's fluid thioglycollate medium (RFTM). Moreover, the same samples were analyzed by 2 separate research groups. The results obtained by PCR showed high sensitivity and good correlation between research groups. In addition, this method is faster than histopathology and incubation on RFTM and less expensive than histopathology. Moreover, nested PCR requires less specialized training for technicians than histology. Histopathology also showed high specificity and a good correlation between research groups. Results from incubation on RFTM suggest that this method could give divergent results between research groups, particularly in the case of low levels of infection, but it is nevertheless useful for disease-monitoring purposes. PCR is appropriate for rapidly screening large numbers of clams.

Perkinsosis in the clams Ruditapes decussatus and R. philippinarum in the Northeastern Atlantic and Mediterranean Sea: A review

“…Histopathology is very informative concerning the tissue location of the parasites, their development stages and the type and extent of associated host response, including tissue damage. However, this technique is less sensitive, more time consuming and expensive than RFTM for detecting the pathogen (Rodríguez and Navas, 1995;Villalba et al, 2005;Balseiro et al, 2010).…”

“…The majority of the primers target ribosomal RNA loci (i.e., NTS, 18S, ITS1, 5.8S, and ITS2). Some PCR assays have been developed specifically to detect P. olseni DNA (De la Herrán et al 2000;Elandaloussi et al, 2004;Robledo et al 2000;Balseiro et al, 2010). A PCR-enzyme-linked immunosorbent assay (ELISA) technique was developed to detect P. olseni DNA, and showed a 100-fold higher sensitivity than visualisation of PCR products on ethidium bromide stained agarose gels (Elandaloussi et al, 2004).…”

Development and applications of Ray’s fluid thioglycollate media for detection and manipulation of Perkinsus spp. pathogens of marine molluscs

“…In addition to the 7-category Mackin scale broadly used with USA oysters and others (Ray, 1954b;Balseiro et al, 2010), there are other scales that alter the range of values or the resolution of the scale or both. For example, Andrews and Hewatt (1957) as well as Ray et al (1953) decreased the resolution of the Mackin scale by compressing all ratings into the ranks of 0, 1, 3, and 5.…”

“…Where separate tissue samples are needed for independent histological, biochemical, or DNA-based assays on RFTM-assayed molluscs (Balseiro et al, 2010), such samples may be excised and preserved before wet weights of RFTM-assayed tissues are recorded.…”

Development and application of a novel histological multichrome technique for clam histopathology

“…Balseiro et al (2010) compared histology to other methods for the detection of Perkinsus sp. in several bivalve species and acknowledged its high reliability, albeit recognizing its considerable technical demands, namely the need of expert researchers, when compared to modern PCR-based diagnostics.…”